Optimization and diagnostic performance of a single multiparameter lineblot in the serological workup of systemic sclerosis

Optimization and diagnostic performance of a single multiparameter lineblot in the serological workup of systemic sclerosis

Journal of Immunological Methods 379 (2012) 53–60 Contents lists available at SciVerse ScienceDirect Journal of Immunological Methods journal homepa...

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Journal of Immunological Methods 379 (2012) 53–60

Contents lists available at SciVerse ScienceDirect

Journal of Immunological Methods journal homepage: www.elsevier.com/locate/jim

Research paper

Optimization and diagnostic performance of a single multiparameter lineblot in the serological workup of systemic sclerosis C. Bonroy a,⁎, J. Van Praet b, V. Smith b, K. Van Steendam c, T. Mimori d, E. Deschepper e, D. Deforce c, K. Devreese a, F. De Keyser b a Laboratory of Clinical Biology, Department of Clinical Chemistry, Microbiology and Immunology, Ghent University Hospital (2P8), De Pintelaan 185, B-9000 Ghent, Belgium b Department of Rheumatology, Ghent University Hospital (0K12), De Pintelaan 185, B-9000 Ghent, Belgium c Laboratory for Pharmaceutical Biotechnology, Ghent University, Harelbekestraat 72, B-9000 Ghent, Belgium d Department of Rheumatology and Clinical Immunology, Kyoto University Graduate school of Medicine, 54 Shogoin-Kawahara-cho, Sakyo-ku Kyoto 606–8507, Japan e Biostatistics Unit, Ghent University, Zwijnaardsesteenweg 314 (Blok F), B-9000 Ghent, Belgium

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Article history: Received 9 September 2011 Received in revised form 1 March 2012 Accepted 1 March 2012 Available online 11 March 2012 Keywords: Anti-extractable nuclear antigen antibodies Systemic sclerosis Anti-RNA-polymerase-III antibodies Anti-PM/Scl antibodies Lineblot Diagnostic accuracy

a b s t r a c t Introduction: Detection of systemic sclerosis-associated antibodies (SSc-Ab) in routine clinical practice is mostly restricted to anti-centromere and anti-topoisomerase-I antibodies. However, also other SSc-Ab (e.g. anti-RNA-polymerase-III, anti-PM/Scl, anti-fibrillarin and antiTh/To) have been shown to be valuable diagnostic and prognostic markers for the disease, but testing methodologies for their detection are laborious and time-consuming. This study aimed to optimize interpretational criteria of a multiparameter lineblot (LB) for the parallel detection of SSc-Ab. We also assessed its global diagnostic value as an alternative for combined conventional techniques (CCT) in the serological workup of systemic sclerosis (SSc) patients. Methods: The presence of SSc-Ab (anti-centromere, anti-topoisomerase-I, anti-RNA-polymeraseIII, anti-PM/Scl, anti-fibrillarin and anti-Th/To) was identified by LB on 145 consecutive SSc patients and on 277 disease controls. Diagnostic sensitivity and specificity were calculated for both individual reactivities and the global LB. Cohen's kappa coefficient was used to examine agreement between LB and CCT and guided the definition of final interpretational criteria for LB. Results: Applying the optimal cut-off values and interpretational criteria, LB identified SSc-Ab in 110 SSc patients (sensitivity= 76%) and in 19 disease controls (specificity = 93%). Globally, there was a substantial agreement between CCT and LB (κ = 0.787, concordance 92.4%). LB and CCT showed a very good correlation (κ > 0.800) for most SSc-Ab (anti-centromere, antitopoisomerase-I, anti-RNA-polymerase-III and anti-PM/Scl). The best agreement for anti-RNApolymerase-III and anti-PM/Scl was achieved when positivity for both components was taken as a criterion. Conclusions: LB is a reliable alternative for the laborious and time-consuming conventional techniques in the diagnostic workup of SSc, especially for the detection of anti-centromere, anti-topoisomerase-I, anti-RNA-polymerase-III and anti-PM/Scl. © 2012 Elsevier B.V. All rights reserved.

Abbreviations: SSc-Ab, systemic sclerosis-associated antibodies; IIF, indirect immunofluorescence; LB, lineblot; CCT, combined conventional techniques; ROC, receiver operator characteristics; SLE, systemic lupus erythematosus, ANA, antinuclear antibody, WB, western blotting; P-IP, protein radio-immunoprecipitation; R-IP, RNA immunoprecipitation; DID, double immunodiffusion ⁎ Corresponding author at: Ghent University Hospital (2P8), De Pintelaan 185, B-9000, Ghent, Belgium. Tel.: + 32 9 332 36 31; fax: + 32 9 332 49 85. E-mail addresses: [email protected] (C. Bonroy), [email protected] (J. Van Praet), [email protected] (V. Smith), [email protected] (K. Van Steendam), [email protected] (T. Mimori), [email protected] (E. Deschepper), [email protected] (D. Deforce), [email protected] (K. Devreese), [email protected] (F. De Keyser). 0022-1759/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.jim.2012.03.001

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1. Introduction Traditionally, systemic sclerosis (SSc) patients with established disease are divided based on the extent of skin thickening into limited cutaneous SSc and diffuse cutaneous SSc; a distinction confirmed by the identification of different SSc-associated antibodies (SSc-Ab) in each subset (LeRoy et al., 1988). Apart from their diagnostic value, these SSc-Ab (e.g. anti-topoisomerase-I (otherwise known as anti-Scl70), anti-centromere, anti-PM/Scl, anti-fibrillarin and antiRNA-polymerase-III antibodies) are of prognostic value due to their association with particular disease manifestations (Ho and Reveille, 2003; Steen, 2005). Recently, there has been major interest in identifying patients in the early phase of the disease. According to LeRoy and Medsger, patients without skin involvement but with Raynaud's phenomenon are diagnosed (and classified) in an early stage as limited SSc based on SSc-type findings on nailfold capillary microscopy and/or positive serology selective for SSc (LeRoy and Medsger, 2001). Koenig et al. confirmed that both criteria are independent predictive factors in the progression of isolated Raynaud phenomenon to SSc (Koenig et al., 2008). The rationale behind the need for timely recognition of SSc is the presence of significant organ involvement in the ‘early’ stage (Van Praet et al., 2011). In these criteria, serology selective for SSc is considered positive if one of the following SSc-Ab were present: anti-centromere, anti-topoisomerase-I, anti-fibrillarin, anti-PM/Scl, anti-RNA-polymerase-III or antifibrillin (LeRoy and Medsger, 2001). Strictly, Th/To antibodies are not included in these criteria, but the cohort study that validated the criteria found anti-Th/To to be predictive for progression from Raynaud's phenomenon to definite SSc (Koenig et al., 2008). Therefore, a modified list of the antibodies is often used in daily clinical practice. Until now, most routine laboratories have only reported test results of anti-centromere and anti-topoisomerase-I completed with the detection of antinucleolar antibodies by indirect immunofluorescence (IIF) (Harvey and McHugh, 1999; Reveille and Solomon, 2003; Walker and Fritzler, 2007). However, a nucleolar pattern on IIF is only indicative for the presence of SSc-Ab (e.g. anti-PM/Scl, anti-Th/To, anti-fibrillarin and anti-RNA-polymerase family). Also other autoantibodies non-specific for SSc (e.g. NOR-90 antibodies) may be responsible for the nucleolar pattern (Fritzler et al., 1995; Fujii et al., 1996). Historically, these SSc-associated nucleolar autoantibodies could only be identified by the use of a combination of laborious and time-consuming conventional techniques which are not applicable in a routine laboratory setting. Nowadays, several immunoassays targeting these individual SSc-Ab are becoming available (Mahler and Raijmakers, 2007; Santiago et al., 2007; Hanke et al., 2009; Mahler and Fritzler, 2009; Satoh et al., 2009; Hanke et al., 2010; Maes et al., 2010; Villalta et al., 2010). In recent years, detection of anti-RNA-polymerase-III antibodies by ELISA has found its way to the routine laboratory (Kuwana et al., 2005; Maes et al., 2010). However, most of these assays are not standardized using variable techniques and antigen sources and it is unclear how they should be applied efficiently in routine laboratory. Recently, a new lineblot immunoassay (LB) for the parallel detection of 12 SSc-Ab has been

developed, but assay validation against clinical diagnosis (including patients with limited SSc) and comparison with the combined conventional techniques (CCT) has not yet been performed. This study aims to optimize and to assess the diagnostic performance of this single multiparameter LB for the parallel detection of SSc-Ab as an alternative for CCT. This was accomplished by a staged approach. First, frequencies for each individual SSc-Ab were evaluated. Second, interpretational criteria were defined, guided by the agreement between LB and CCT. Finally, a global evaluation of the accuracy of the LB in the diagnosis of SSc was performed. 2. Methods 2.1. Samples A total of 422 serum samples were used to define disease and control groups for receiver operator characteristics (ROC) analysis. The disease group consisted of 145 consecutive SSc patients fulfilling LeRoy and Medsger criteria for SSc and was divided into limited SSc (n= 41), limited cutaneous SSc (n= 84) and diffuse cutaneous SSc (n= 20) (LeRoy et al., 1988; LeRoy and Medsger, 2001). Seventy-seven percent were female and the mean± SD age was 54 ± 13 years. The mean± SD duration of Raynaud's phenomenon was 10± 12 years. One hundred forty-four patients were Caucasians, whereas one was from North-African origin. Further demographic and clinical characteristics of the patients had been described earlier (Van Praet et al., 2011). The controls consisted of 277 patients with established clinical diagnosis and represented the following connective tissue diseases (classified according to ACR criteria where applicable): 90 patients with rheumatoid arthritis, 58 patients with systemic lupus erythematosus (SLE), 50 patients with spondyloarthropathy, 49 patients with osteoarthritis, 18 patients with polymyalgia rheumatica and 12 patients with ANCA-associated vasculitis (Tan et al., 1982; Arnett et al., 1988; Fries et al., 1990; Altman, 1991). This study was conducted after approval by the Ethics Committee of the Ghent University Hospital and all SSc patients signed informed consent. Control samples used for this study were from the hospital serum bank and were obtained mostly in the context of previously reported studies (Hoffman et al., 2002; De Rycke et al., 2004; Van Praet et al., 2009). 2.2. Analytical methods 2.2.1. Antinuclear antibody (ANA) detection IIF on HEp-2000 cells was performed according to the manufacturer's instructions (Immunoconcepts, Sacramento, CA, USA), using a serum dilution of 1:40 in order to obtain high sensitivity. 2.2.2. Western blotting with a nuclear extract Western blotting (WB) was performed as previously described (Van Praet et al., 2011). In brief, a nuclear extract from K562 cells was separated on a 10% SDS-PAGE and subsequently blotted on nitrocellulose membranes. After overnight incubation with prediluted serum (1:100), antibody binding was visualized using HRP-labelled goat anti-human IgG in combination with enhanced electrochemiluminescent

C. Bonroy et al. / Journal of Immunological Methods 379 (2012) 53–60

detection substrate (Supersignal West Dura Extended Duration Substrate, Pierce, Rockford, IL, USA), using the Versadoc Imaging System (Biorad). 2.2.3. Protein radio-immunoprecipitation with a total cell extract Protein radio-immunoprecipitation (P-IP) was performed as previously described (Van Praet et al., 2011). In brief, K562 cells were cultured overnight in methionine- and cystinedeficient RPMI (Sigma, St. Louis, MO), supplemented with 35S-methionine and 35S-cystine (Perkin Elmer, Wellesley, MA, USA). A total cell extract of these labelled cells was used for immunoprecipitation with protein A-sepharose beads (protein A–Sepharose beads, Sigma) bound to patient's IgG. After washing, precipitated proteins were fractionated by 8% SDS-PAGE and visualized by autoradiography. 2.2.4. INNO-LIA™ ANA Update line immunoassay The INNO-LIA ANA Update (LIA) (Innogenetics NV, Zwijnaarde, Belgium) was performed as previously described (Meheus et al., 1999; Pottel et al., 2004). This assay contains the following antigens: SmB (recombinant), SmD (synthetic peptide), RNP-A (recombinant), RNP-C (recombinant), RNP70 k (recombinant), Ro52/SSA (recombinant), Ro60/SSA (natural purified), SSB (recombinant), centromere-B (recombinant), topoisomerase-I (recombinant), Jo-1 (recombinant), ribosomal P (synthetic peptide), and histones (natural purified). Each line of the test strip was compared with the respective line on a reference strip, obtained by testing a cut-off control sample in each run. 2.2.5. RNA immunoprecipitation with a total cell extract RNA immunoprecipitation (R-IP) was performed as previously described (Fujita et al., 2008; Van Praet et al., 2011). In brief, patient's IgG coupled to protein A-sepharose beads were incubated with a total HeLa cell extract. After washing, precipitated RNAs were extracted with phenol/chloroform/ isoamyl alcohol, separated on a 7 M urea-10% PAGE and visualized with silverstaining. 2.2.6. Double immunodiffusion with topoisomerase-I antigen The double immunodiffusion test (DID) was performed using the NOVA Gel Scl-70 S test system (Inova Diagnostics Inc., San Diego, United States of America). Briefly, patient serum samples in two serum dilutions (1:1 and 1:4) and topoisomerase-I antigen preparation (purified from calf thymus extract) (Inova Diagnostics Inc., San Diego, United States of America) were allowed to diffuse passively through a gel support matrix. When antibodies targeting topoisomerase-I were present, a visible precipitin line was formed in the gel at the point of antigen-antibody equivalence. Confirmation of the topoisomerase-I activity in the patient's serum was made if its precipitin line characteristically merged with the line formed by a known control sample. 2.2.7. Lineblot assay The Systemic Sclerosis (Nucleoli) Profile Euroline (IgG) lineblot assay (LB) was applied according to the manufacturer's instructions (EUROIMMUN AG, Lübeck, Germany). This assay contains the following antigens: human recombinant centromere-A, centromere-B, RNA-polymerase-III/RP11

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(RNA-polymerase-III subunit POLR3K), RNA-polymerase-III/ RP155 (RNA-polymerase-III subunit POLR3A), PM/Scl-75 (75 kDa protein), PM/Scl-100 (100 kDa protein), Fibrillarin, Th/To, NOR90, Ku and Ro52 and native purified topoisomerase-I. The following recombinant proteins were obtained by expressing their corresponding human cDNA with the baculovirus system in insect cells: centromere-A, centromere-B, PM/Scl-75 (75 kDa protein), PM/Scl-100 (100 kDa protein), Ku and Ro52. The other recombinant proteins (e.g. RNA-polymerase-III/RP11, RNA-polymerase-III/ RP155, fibrillarin, Th/To and NOR90) were obtained in an E.coli expression system. All LB assays were performed in an automated way, using the EUROblotmaster (EUROIMMUN AG, Lübeck, Germany). In short, the prediluted samples (1:101) were incubated with the lineblot strip for 30 min. Binding of specific autoantibodies was visualized with goat anti-human IgG labelled with alkaline phosphatase in combination with the chromogen substrate NBT/BCIP. Results were digitized using a calibrated flatbed scanner, and absolute signal intensities were imported by a computer program for further analysis (EUROLineScan, EUROIMMUN AG). Nonconverted scanned signal strengths obtained for each SScAb were used for data analysis. This manuscript focuses on the SSc-Ab included in the classification criteria for SSc (see introduction) (LeRoy and Medsger, 2001). Therefore, only the following SSc-Ab are taken into account: anticentromere, anti-topoisomerase-I, anti-RNA-polymerase-III, anti-PM/Scl and anti-fibrillarin. Based on the results of Koenig et al. we added anti-Th/To to the list (Koenig et al., 2008). Anti-fibrillin was not included, as these antibodies were not detectable by the LB. Data on the non-SSc-specific autoantibodies (e.g. anti-Ku, anti-Ro52 and anti-NOR90) were excluded from further analysis. 2.2.8. Combined conventional techniques In order to define analytical ‘true’ positivity for the SScAb, all SSc patients (n = 145) were analysed with the following conventional techniques: ANA detection by IIF on HEp2000 cells, WB with a nuclear extract, P-IP with a total cell extract and DID. In addition, R-IP was performed on the IIF positive samples in which no other ANA identification techniques (including results of LIA and CCT) yielded a specific ANA reactivity (n = 17), and on all samples with a nucleolar fluorescence pattern that could not be explained by the presence of PM/Scl, Topo-I or RNA-polymerase III antibodies (n = 4). 2.2.9. Statistical analysis For evaluation of the LB performance characteristics, ROC curves on the obtained signal strengths (U) for each individual antigen were constructed (Greiner et al., 2000). Diagnostic sensitivity and specificity were evaluated for the individual reactivities using the cut-off values proposed by the manufacturer (>10U positive). These cut-off values were further used for dichotomization of the original continuous data. After dichotomization of the data, identification of the SSc-Ab by LB was compared with their identification by CCT. Agreement of CCT versus LB was assessed by contingency tables and kappa statistics for each reactivity (Cohen, 1960). Different combinations of the components of centromere (centromere-A and/or centromere-B), RNA-polymerase-III

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(RNA-polymerase-III/RP11 and/or RNA-polymerase-III/RP155) and PM/Scl (PM/Scl-75 and/or PM/Scl-100) antibodies were evaluated. Next, interpretational criteria for LB with regard to different combinations of the components of centromere, RNA-polymerase-III and PM/Scl antibodies were defined based on the highest kappa-agreement. In the final analysis, these defined interpretational criteria were used to analyse the global LB performance characteristics. For comparison of proportions, Chi-square testing with Yates' correction for continuity was applied. Two-sided p-values b 0.05 were considered significant. Statistical analysis was performed with PasW 18.0 statistical package (SPSS Inc., Chicago, IL, USA). 3. Results 3.1. Frequencies of the SSc-Ab detected by LB For evaluation of the LB performance characteristics, ROC curves on the obtained signal strengths for the individual reactivities were constructed. Area under the curve values are shown in Table 1. Our data showed overlapping distributions of patients and controls for anti-RNA-polymerase-III, anti-PM/Scl, anti-Th/To and anti-fibrillarin (0.5 included in 95% the confidence intervals of the area under the curves). Using the cut-off values proposed by the manufacturer (signal strength >10U), we determined the frequencies of SSc-Ab detected by LB in patients (n = 145) and controls (n = 277) (see Table 1). The distributions of signal strengths for the different SSc-Ab on LB in SSc patients and controls are available as supplementary data (see Appendix 1). Globally, 58 (21%) of the controls showed response signals >10U for minimal one SSc-Ab. A minority (n= 7) of these samples reacted with several antigens: 2 samples with centromere-A and centromere-B, 1 sample with centromere-A, PM/Scl-75 and PM/Scl-100, 1 sample with centromere-B and PM/Scl-75, 1 sample with topoisomerase-I and PM/Scl-100, 1 sample

with RNA-polymerase-III/RP155 and PM/Scl-100 and 1 sample with PM/Scl-75 and Th/To. 3.2. Definition of interpretational criteria guided by the correlation between LB and CCT We next compared the results of CCT and LB in order to define final interpretational criteria. The results obtained by LB were compared with the ones acquired by CCT. To this end, all 145 SSc patients were categorized as ‘CCT positive’ or ‘CCT negative’ based on the combination of results obtained by the different conventional techniques. CCT results were considered positive if the criterion for positivity was fulfilled as defined in Table 2. By means of CCT, SSc-Ab were detected in 113 (78%) of the 145 SSc patients previously described (Van Praet et al., 2011). The final interpretational criteria were based on the highest kappa-agreement between CCT and LB (see 2.2.8. statistical analysis). For anti-centromere, the best kappa-agreement was obtained when either reactivity for centromere-A or centromere-B were above the cut-off value (anti-centromere-A or anti-centromere-B> 10U). For anti-RNA-polymerase-III, combined positivity for both antiRNA-polymerase-III/RP11 and anti-RNA-polymerase-III/RP155 was selected as a criterion (anti-RNA-polymerase-III/RP11 and anti-RNA-polymerase-III/RP155 >10U). Similarly, combined positivity for both PM/Scl-75 and PM/Scl-100 achieved the highest kappa-agreement (anti-PM/Scl-75 and anti- antiPM/Scl-100 > 10U). There was an almost perfect agreement (κ > 0.800) between CCT and LB for anti-centromere, antitopoisomerase-I, anti-RNA-polymerase-IIII and anti-PM/Scl. In contrast, for Th/To antibodies, only weak agreement (κ = 0.146) was obtained. No statistics on fibrillarin antibodies for the comparative analysis between LB and CCT was computed as none of the SSc serum samples was found positive on CCT. An overview of the results is given in Table 3. 3.3. Global performance characteristics of the optimized LB compared to CCT

Table 1 ROC analysis results and frequencies of SSc-Ab in patients and controls.

Anti-CENP-A Anti-CENP-B Anti-topo-I Anti-RP11 Anti-RP155 Anti-PM/ Scl-75 Anti-PM/ Scl-100 Antifibrillarin Anti-Th/To

AUC (95%CI)

Total SSc patients* n=145

Total controls** n=277

0.672 0.691 0.598 0.508 0.495 0.500

66 64 28 12 14 13

6 (2.2) 3 (1.1) 6 (2.2) 4 (1.4) 7 (2.5) 22 (7.9)

(0.611–0.733) (0.630–0.753 (0.540–0.657) (0.448–0.568) (0.435–0.554) (0.441–0.559)

(46) (44) (19) (8.3) (9.7) (9.0)

0.574 (0.514–0.634)

15 (10.3)

11 (4.0)

0.474 (0.415–0.532)

2 (1.4)

0 (0.0)

0.496 (0.439–0.553)

3 (2.1)

6 (2.2)

*Findings are reported as number of positive patients (% of positivity within patients). **Findings are reported as number of positive controls (% of positivity within controls). Anti-CENP-A = anti- centromere-A; anti-CENP-B = anti-centromere-B; antitopoisomerase-I = anti-topo-I, anti-RP11 = anti-RNA-polymerase III subunit POLR3K; anti-RP155 = anti-RNA-polymerase III subunit POLR3A; anti-PM/ Scl-75 = anti-PM/Scl 75 kDa protein; anti-PM/Scl-100 = anti-PM/Scl 100 kDa protein.

To evaluate the overall diagnostic performance of the LB we applied the above described interpretational criteria on the SSc population and compared the results with the ones obtained by CCT. For this analysis, samples were considered ‘LB positive’ if at least one of the SSc-Ab was found positive on LB (as defined supra 2.2.6. Lineblot assay). Table 2 Definition of positivity for SSc-Ab on CCT. Antigens

Definition of CCT positive

Number of sera

Centromere Topoisomerase-I RNA-polymerase III

Positive for centromere with IIF or WB Positive for topoisomerase-I with DID Positive for RNA-polymerase III with P-IP Positive for PM/Scl with WB or P-IP and DID negative Positive for fibrillarin with P-IP Positive for Th/To on R-IP

n = 72 n = 26 n = 11

PM/Scl Fibrillarin Th/To

n=4 none n=4

CCT = combined conventional techniques; IIF = Indirect Immunofluorescence; P-IP= protein immunoprecipitation; WB = western blotting with nuclear extract; LIA= line immune assay INNO-LIA™ ANA Update; R-IP= RNA-Immunoprecipitation; DID = double immunodiffusion with Topoisomerase-I antigen.

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Table 3 Comparison between CCT and LB for each individual SSc-Ab and selection of interpretational criteria. Comparison LB versus CCT

LB performance characteristics

Total SSc patients (n = 145)

Total cohort (n = 422)

CCT + * % (n/145)

SSc-Ab and combinations **

κ

Sensitivity % (n/145)

Specificity %

Anti-CENP

50 (72)

Anti- Topo-I Anti-RNA-PIII

18 (26) 7.6 (11)

Anti-PM/Scl

2.8 (4)

Anti-Th/To

2.7 (4)

CENP-A or -B CENP-A and -B Topo-I RNA-PIII/RP11 or RP155 RNA-PIII/RP11 and RP155 PM/Scl-75 or PM/Scl-100 PM/Scl-75 and PM/Scl-100 Th/To

0.848 0.820 0.909 0.831 1.000 0.167 0.854 0.146

49 (71) 41 (59) 19 (28) 10 (15) 7.6 (11) 17 (25) 2.1 (3) 2.1 (3)

97 > 99 98 96 100 88 > 99 98

*CCT + = combined conventional techniques positive as defined in Table 2. **Selected interpretational criteria are marked in bold (combination with highest kappa-agreement). anti-CENP-A = anti-centromere-A; anti-CENP-B = anti-centromere-B; anti-topoisomerase-I = anti-topo-I. anti-RNA-PIII = anti-RNA-polymerase III, anti-RP11 = anti-RNA-polymerase III subunit POLR3K; anti-RP155 = anti-RNA-polymerase III subunit POLR3A; anti-PM/ Scl-75 = anti-PM/Scl 75 kDa protein; anti-PM/Scl-100 = anti-PM/Scl 100 kDa protein.

We found an overall diagnostic sensitivity of 76% and a specificity of 93%. The contingency table is shown in Table 4a. SSc-Ab were detected in 110 SSc patients by LB: anti-centromere (anti-centromere-A or anti-centromere-B >10U) in 71 (49%), anti-topoisomerase-I (>10U) in 28 (19%), anti-RNA-polymerase-III (anti-RNA-polymerase-III/ RP11 and anti-RNA-polymerase-III/RP155 >10U) in 11 (7.6%), anti-PM/Scl (PM/Scl-75 and PM/Scl-100 > 10U) in 3 (2.1%), anti-fibrillarin (>10U) in 2 (1.4%), anti-Th/To (>10U) in 3 (2.1%) (see sensitivity Table 3). Most patient samples show single SSc-Ab reactivity on lineblot (n = 101/110; 92%). Nineteen control samples tested positive on LB for following antigens: 6 for Th/To, 6 for topoisomerase-I, 3 for centromere-A only, 1 for centromere-B only, 2 for combined centromere-A and centromere-B and 1 for combined centromere-A and PM/

Table 4 Contingency tables describing (A) the global diagnostic performance using the proposed interpretational criteria and (B) the comparison of CCT versus LB. 4A. All patients (n = 422)

Non-SSc controls SSc patients Sensitivity = 76%; specificity = 93%

LB* −

+

258 35

19 110

4B. SSc patients (n = 145)

LB* −

CCT + ** 7 CCT − 28 Concordance = 92.4%; κ = 0.787; 95%CI = 0.618–0.884

+ 106 4

*A sample was considered ‘LB positive’ if at least one out of the SSc-Ab was positive using the interpretational criteria: anti-CENP-A or anti- CENP-B >10U, anti-Topo-I > 10U, anti-RNA-polymerase-III/RP11and anti-RNApolymerase-III/RP155 >10U, anti-PM/Scl-75 and anti-PM/Scl-100 > 10U, anti-Th/To > 10U, anti-fibrilarin > 10U. **A sample was considered ‘CCT positive’ if at least one of the SSc-Ab was positive on the conventional techniques as defined in Table 2. Sera not fulfilling these criteria were classified as ‘CCT negative’.

Scl. Most of the positive control sera were derived from SLE patients (n= 9/58, 16% within SLE patients) and osteoarthritis patients (n= 4/49, 8% within osteoarthritis patients). The contingency table assessing global agreement of CCT versus LB is shown in Table 4b. Globally, there is a substantial agreement (κ = 0.787, concordance 92.4%) between the CCT and LB. Four LB positive sera could not be confirmed by CCT. Two of these samples were positive for anticentromere, not showing the specific centromere pattern on IIF. The other samples were positive for anti-Th/To (high signal intensity, n = 1) or anti-topoisomerase-I (low intensity signal, n = 1). Inversely, 7 CCT positive sera were missed on LB. Four of these samples corresponded with the following reactivities on CCT: anti-topoisomerase-I (n = 1), anti-Th/To (n = 2) and anti-PM/Scl (n = 1). CCT also identified 3 additional anti-centromere sera. These samples were only positive with one of both reference techniques (2 samples IIF positive, 1 sample WB positive (only CENP-C band visible)). Remarkably, 4 of the 7 sera missed by LB were derived from patients belonging to the lSSc subset (n = 41). In contrast, 1 of the 7 sera missed by LB belonged to the lcSSc subset (n = 84) and 2 to the dcSSc subset (n = 20). Nevertheless, the tendency toward less sensitive detection of SSc-Ab in lSSc patients using LB (n = 30/41) compared to CCT (n = 32/41) was not significant (p = 0.7977). 3.4. Comparison of LB with LIA for the detection of anticentromere and anti-topoisomerase-I InnoLIA ANA is an extensively used assay for the detection of anti-topoisomerase-I and anti-centromere-B antibodies and has been compared with the conventional techniques in a large multicenter study (Meheus et al., 1999). Therefore, we also compared the results obtained by LB with the results obtained by LIA. For anti-centromere-B and anti-topo-I, kappa values of 0.888 and 0.885 were found respectively. Cross tabulations are shown in Appendix 2. 4. Discussion Routine algorithms for ANA identification lack sensitivity for the detection of SSc-Ab and should be supplemented

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with additional assays that detect anti-RNA polymerase-III, anti-PM/Scl and anti-Th/To (Van Praet et al., 2011). Nowadays, several immunoassays targeting these SSc-Ab are commercially available as a promising alternative for the laborious CCT (Santiago et al., 2007; Maes et al., 2010; Villalta et al., 2010). Most immunoassays use highly purified recombinant antigens immobilized on a solid phase. In contrast, conventional techniques employ native antigens, allowing better detection of antibodies directed against conformational-dependent epitopes as well as epitopes originating from post-translational modifications (Phan et al., 2002; Albon et al., 2011). Due to this difference in antigen sources, discordant results in validation studies are selfevident (Satoh et al., 2009; Meyer et al., 2010; Jaskowski et al., 2011). A second factor that influences the difference between methods is the defined cut-off value (Albon et al., 2011). Correct positioning of the cut-off values is essential for obtaining the most optimal balance between sensitivity and specificity (Greiner et al., 2000). In our study, we presumed the conventional techniques to be the ‘golden standard’, as they were historically used to characterize the SScAb and their associated clinical manifestations (Reimer et al., 1987; Reimer et al., 1988; Okano et al., 1993; Chang et al., 1998; Van Eenennaam et al., 2002). After optimization of the interpretational criteria of a single multiparameter LB, we investigated SSc specific serological profiles in a consecutive cohort in parallel by LB and CCT. Interpretational criteria were selected based on the most optimal agreement with the ‘golden standard’ tests. As all three disease subsets (limited SSc, limited cutaneous SSc and diffuse cutaneous SSc) were included in the cohort, we were able to evaluate performance of the LB in both SSc patients with skin involvement and those without. Our data confirm that positive test results for anticentromere and topoisomerase-I are highly specific for SSc (Russo et al., 2000; Ho and Reveille, 2003; Tamby et al., 2007; Hanke et al., 2010; Maes et al., 2010). An almost perfect agreement between LB and CCT can be observed for both parameters. For anti-RNA-polymerase-III, the obtained signal intensities for patient and controls showed overlapping distributions. Nevertheless, when positivity for both RNApolymerase-III components was selected as a criterion, a good discrimination was obtained between patients and controls, and there was a perfect agreement between LB and CCT. In parallel, we retained acceptable sensitivity (7.6%) comparable with the previously reported data on Caucasian patients with SSc (Koenig et al., 2008; Maes et al., 2010; Meyer et al., 2010). Anti-PM/Scl antibodies are associated with SSc, polymyositis, and the polymyositis/scleroderma overlap syndrome (Mahler and Fritzler, 2009). Similar to RNA-polymerase-III antibodies, our data showed for anti-PM/Scl overlapping distributions in signal intensities of patient and controls. These data were compatible with the observation that low reactivity to PM1-alpha antibodies was seen in disease controls (Maes et al., 2010). When positivity for either anti-PM/Scl75 or anti-PM/Scl-100 was used as a criterion, we found 17% positivity for SSc patients with a parallel specificity of 88%. In parallel, a low agreement between LB and CCT for PM/Scl antibodies was found (k = 0.167). As most optimal agreement with CCT was a priority, we finally selected

combined positivity for both components as a criterion (k = 0.854). Positive impact was seen on clinical specificity (>99%). Using this interpretational criterion, we retained three positive SSc patients (2.1%). This percentage is comparable with the prevalence found in the Pittsburgh Scleroderma database, but is somewhat lower than more recently published results (Steen, 2005; Maes et al., 2010). It has been suggested that variations in the presence of anti-PM/ Scl might be related to geo-ethnic, racial and genetic differences (Genth et al., 1990; Oddis et al., 1992). Anti-Th/To antibodies are present in about 2–5% of the SSc patients, being probably more prevalent in Japanese patients (Jacobsen et al., 1998). Our CCT confirm that anti-Th/To are relatively common (19%, n = 4/21) when a serum selection bias was applied based on ANA IIF pattern and the presence of other SSc-Ab (Van Praet et al., 2011). However, our data cannot be compared directly with the data obtained by Cerebelli et al. (Ceribelli et al., 2010). This group performed R-IP on a selection of samples that was negative for anticentromere, anti-topoisomerase-I antibodies and anti-RNA polymerase III (n = 41). Within this selection of samples, 14 showed nucleolar staining on IIF. Anti-Th/To was identified in 57% (n = 8/14) of the final selection (Ceribelli et al., 2010). The presence of anti-Th/To (tested by P-IP) is also described in SLE, polymyositis, primary Raynaud and even in healthy donors (Kuwana et al., 2002). Our LB data confirm overlapping distributions for patients and controls. When a cut-off value of 10U was applied, 3 patient samples (2.1%) and 6 control samples (2.2%) were found positive. Our data showed a low agreement between LB and CCT for anti-Th/ To. Moreover, the signal intensity on LB was not indicative for confirmation on R-IP (data not shown). Our results suggest that the detection of Th/To antibodies by P-IP and LB might be different stories. However, due to low frequency of anti-Th/To positive samples positive in our study, no strong conclusions regarding this antibody could be drawn. By CCT, none of the SSc serum samples were positive for anti-fibrillarin antibodies, despite the ability of our reference tests to detect fibrillarin antibodies (a positive control showed a band at 34kD with WB and P-IP, data not shown). Anti-fibrillarin antibodies are generally accepted to be a marker very specific marker for SSc and are detected more frequently among African American patients with SSc compared to other ethnic groups, probably explaining the lack of anti-fibrillarin CCT positive patients in our Caucasian cohort (Sharif et al., 2011; Van Praet et al., 2011). In contrast, anti-fibrillarin antibodies were identified by LB in the disease population at low frequency (2.1%) using a cut-off value of 10U. However, as our data on anti-fibrillarin showed overlapping distributions between patients and controls and all three positive patient samples showed low signal intensity on LB, conclusions regarding this antibody are limited. Globally, when LB is implemented according to the proposed criteria, SSc-Ab were found in 76% of the SSc patients. In parallel, a diagnostic specificity of 93% and a substantial agreement between LB and CCT were found. Most samples show single reactivity, confirming that SSc-Ab are serologically independent and mutually exclusive autoantibodies (Steen, 2005). In sum, we propose interpretational criteria for a multiparameter LB. In such a format, LB is a reliable alternative

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for the laborious and time-consuming conventional techniques in the diagnostic workup of SSc, especially for the detection of anti-centromere, anti-topoisomerase-I, anti-RNApolymerase III and anti-PM/Scl. To detect anti-Th/To and anti-fibrillarin, the LB offers no all-embracing alternative to CCT. However, these reactivities are rarely identified. Conflict Of Interest Statement The authors have declared no conflicts of interest. Funding Statement Jens T Van Praet and Katleen Van Steendam are supported by a research grant from the Fund for Scientific Research, Flanders. Supplementary data related to this article can be found online at doi:10.1016/j.jim.2012.03.001. Acknowledgements The technical assistance of Ms Virgie Baert, Ms Annette Heirwegh, Ms Vicky Mortier and Ms An De Saar is greatly acknowledged. References Albon, S., Bunn, C., Swana, G., Karim, Y., 2011. Performance of a multiplex assay compared to enzyme and precipitation methods for anti-ENA testing in systemic lupus and systemic sclerosis. J. Immunol. Methods 365, 126. Altman, R.D., 1991. Classification of disease: osteoarthritis. Semin. Arthritis Rheum. 20, 40. Arnett, F.C., Edworthy, S.M., Bloch, D.A., McShane, D.J., Fries, J.F., Cooper, N.S., Healey, L.A., Kaplan, S.R., Liang, M.H., Luthra, H.S., et al., 1988. The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis. Arthritis Rheum. 31, 315. Ceribelli, A., Cavazzana, I., Franceschini, F., Airo, P., Tincani, A., Cattaneo, R., Pauley, B.A., Chan, E.K., Satoh, M., 2010. Anti-Th/To are common antinucleolar autoantibodies in Italian patients with scleroderma. J. Rheumatol. 37, 2071. Chang, M., Wang, R.J., Yangco, D.T., Sharp, G.C., Komatireddy, G.R., Hoffman, R.W., 1998. Analysis of autoantibodies against RNA polymerases using immunoaffinity-purifed RNA polymerase I, II, and III antigen in an enzyme-linked immunosorbent assay. Clin. Immunol. Immunopathol. 89, 71. Cohen, J.A., 1960. A coefficient of agreement for nominal scales. Educ. Psychol. Meas. 37. De Rycke, L., Peene, I., Hoffman, I.E., Kruithof, E., Union, A., Meheus, L., Lebeer, K., Wyns, B., Vincent, C., Mielants, H., Boullart, L., Serre, G., Veys, E.M., De Keyser, F., 2004. Rheumatoid factor and anticitrullinated protein antibodies in rheumatoid arthritis: diagnostic value, associations with radiological progression rate, and extra-articular manifestations. Ann. Rheum. Dis. 63, 1587. Fries, J.F., Hunder, G.G., Bloch, D.A., Michel, B.A., Arend, W.P., Calabrese, L.H., Fauci, A.S., Leavitt, R.Y., Lie, J.T., Lightfoot Jr., R.W., et al., 1990. The American College of Rheumatology 1990 criteria for the classification of vasculitis. Summary. Arthritis Rheum. 33, 1135. Fritzler, M.J., von Muhlen, C.A., Toffoli, S.M., Staub, H.L., Laxer, R.M., 1995. Autoantibodies to the nucleolar organizer antigen NOR-90 in children with systemic rheumatic diseases. J. Rheumatol. 22, 521. Fujii, T., Mimori, T., Akizuki, M., 1996. Detection of autoantibodies to nucleolar transcription factor NOR 90/hUBF in sera of patients with rheumatic diseases, by recombinant autoantigen-based assays. Arthritis Rheum. 39, 1313. Fujita, Y., Fujii, T., Nakashima, R., Tanaka, M., Mimori, T., 2008. Aseptic meningitis in mixed connective tissue disease: cytokine and anti-U1RNP antibodies in cerebrospinal fluids from two different cases. Mod. Rheumatol. 18, 184. Genth, E., Mierau, R., Genetzky, P., von Muhlen, C.A., Kaufmann, S., von Wilmowsky, H., Meurer, M., Krieg, T., Pollmann, H.J., Hartl, P.W., 1990.

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